Part:BBa_K4674012
The exprssing cassette for tetrahedral ssDNA with cis-auto splicing motif
This cassette is designed for expressing tetrahedral ssDNA with cis-auto splicing motif through flanking with RCORI-105 and RCORI-65 motifs as start and stop points for RepA recognization.
Over the past few years, the swift advancement of DNA nanotechnology has brought forth fresh perspectives for the detection and therapy of cancer. These nanostructures, assembled from single-stranded DNA, exhibit several features that make them suitable candidates for innovative cancer detection strategies.
One of the main advantages of DNA tetrahedron-based approaches in cancer detection is their high programmability and specificity. These structures can be precisely designed to carry various functional components, including ligands. This allows us to create tailored probes capable of targeting specific cancer-related biomarkers with a high degree of accuracy. Additionally, the DNA tetrahedron's inherent stability and rigidity contribute to the stability of the detection platform. This stability is essential for maintaining the integrity of the assembled nanostructure during various stages of the detection process, including sample preparation, target binding, and signal readout.
In summary, DNA tetrahedron structures offer a promising platform for the development of innovative cancer detection methods. Their programmability, versatility, and ability to carry multiple functional components make them well-suited for designing highly sensitive and specific assays that hold the potential to contribute significantly to improved cancer detection and patient outcomes.
Deoxyribozyme: the cis-auto and trans-auto splicing of ssDNA
Deoxyribozyme are DNA molecules habor catalytic activity. Since the discovery in 1994, Deoxyribosomes are proven to cleave RNA phosphoester bond and mediate DNA phosphorylation, adenylation, deglycosylation and ligation. Recently research indicated that DNA can directly self-hydrolyze DNA phosphodiester bonds by forming speicifc structure and using zinc ion as a cofactor.
There are two classes of deoxyribozymes: Class I deoxyribozyme contains 15 conserved nucleotides within a loop region flanked by one or two base-paired stems. The class I deoxyribozyme recognizes substrate sequences of GTTGAAG and hydrolyze the phosphodiester bond between the dinucleotide ApA. The class II deoxyribozyme arries 32 nucleotides within an unpaired bulge that is flanked by two base-paired stems (Gu et al. 2013). To apply the deoxyribozymes to auto-splicing of ssDNA, we selected the classI-R1 sequence for cis-auto splicing and class1-R3 for trans-auto splicing.
Fig. The secondary structure of cis-auto (left) and trans-auto (right) motifs.
The design of DNA tetrahedron assembled from trtrahedral ssDNA
The folding pathway of the tetrahedral ssDNA is illustrated in the figure below. Among six edges, five are composed of 21 bp double helix. The last edge is a “twin double helices” to compensate the necessary of reverse polarity of complementary DNA strands. The 5’ terminus of tetrahedral ssDNA starts at one endpoint, and the PBSII and Zif268 binding motifs are located at the farest two edges, respectively (Conrado, R. J., et al. 2011). The PBSII and Zif268 binding motifs are applied to anchor tetrahedrons on the wall of main chamber in CTC-FAST device though interaction with PBSII and Zif268 proteins.
Since the commercial production of tetrahedral ssDNA is hampered by its highly complementary sequence, we decided to apply the RCR mechanism to gererate the tetrahedral ssDNA (see below section for details). The products from RCR will be circular ssDNA, therefore, we decided to fuse the cis-auto splicing sequence at the 5’ terminus tetrahedral ssDNA. After auto-splicing, the RCR generated circular ssDNA will fold into tetradron, and a 18 nucleotides overhang, which are the residues after splicing, will left at 3’ terminus. The 3’ terminus of FA-conjugated adaptor ssDNA will be complement to this overhang, and the 5’ terminus will contain the trans-auto splicing sequence. Finally, the exogenous ssDNA complementary to the trans-auto slicng sequence could be added to cleave the adaptor ssDNA and release the captured CTCs.
Fig. The design design of DNA tetrahedron assembled from tetrahhedral ssDNA.
References
Conrado, R. J., et al.(2012). DNA-guided assembly of biosynthetic pathways promotes improved catalytic efficiency. Nucleic acids research, 40(4), 1879–1889.
Hongzhou Gu et al. (2013) Small, highly-active DNAs that hydrolyze DNA. J Am Chem Soc. 135(24): 9121–9129.
Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Illegal EcoRI site found at 212
- 12INCOMPATIBLE WITH RFC[12]Illegal EcoRI site found at 212
Illegal NheI site found at 290 - 21INCOMPATIBLE WITH RFC[21]Illegal EcoRI site found at 212
- 23INCOMPATIBLE WITH RFC[23]Illegal EcoRI site found at 212
- 25INCOMPATIBLE WITH RFC[25]Illegal EcoRI site found at 212
Illegal NgoMIV site found at 64
Illegal NgoMIV site found at 271
Illegal NgoMIV site found at 479 - 1000COMPATIBLE WITH RFC[1000]
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